Patient Derived Xenograft & Patient Derived OrganoidsPatient Derived Xenograft & Patient Derived Organoids Flashcards
Modeling tumour biology can be completed using which 2 in vitro pre-clinical laboratory cancer models?
– 2D cancer cell line cultures
– Patient derived organoids “PDO”
How are 2D cancer cell line cultures generated?
2D immortalized clonal cell lines are traditionally grown in a monolayer
- The oldest human cell line was derived from cervical cancer cells (HeLa cells) in 1951
- NCI-60 Cell Line screen (1990)- 60 cell lines represented various cancers, and screened various drugs to inform new drug discovery in multiple cancer types
Why use 2D in vitro models?
• Banks of cell lines allow access to many genotypic
backgrounds of tumours from different tissue types
- Genomic, epigenetic and transcriptomic characterisation
- Suitable for High throughput drug screening
- Have been shown to faithfully recapitulate a number of specific aspects of cancer biology
- Screening of cell lines have been used to identify therapeutic biomarkers
What are the limitations of 2D in vitro models?
Unable to fully capture tumour heterogeneity to directly predict responses in the clinic
Monolayer culture:
• Limited cell-cell contact.
• Limited complex interactions with adjacent cells • 3D complexity is lost
Change in morphology:
• Lack of relevant environmental cues (matrix)
What is an ideal in vitro model?
Suitable for culture
- Ability to proliferate and expand
- Stability in culture
Relevance to patients
- Presence of all cell types within a tumour
- Genotypic characteristics
- Recapitulate architecture of original tissueAnalysis
- Medium/ high throughput
- Reproducibility
- Advanced technological screening
What is an organoid?
A structure resembling the organ from which it is derived
• Cultured in 3D
• Organ-specific cell
types
• Self organizing
• Capable of continuous
expansion in vitro
How are 3D organoid models developed?
E.g Isolating Intestinal crypt
+ growth factors
+ supporting matrix
Sustainable growth by mimicking the intestinal environment
- hydrogel to maintain 3D integrity
- Growth factor supplements to support stem cells
How are tumour organoid models developed?
Growth conditions further adapted to culture material from patient tumours
Tissue from patient biopsies
Dissection, digestion, trituration
Isolated cell fragments from tissue
3D culture in matrix
Optimised media conditions
Organoids have been derived from multiple tissue types
True or false
True
What are the advantages of PDOs as a pre-clinical tool?
Morphology
• Similar histology to
matching patient tumours
• Retain similar cell signaling to native tissue due to 3D architecture
Genetic representation of tumours
• Have the capacity to maintain heterogeneity of tumours as analyzed by DNA sequencing
- Protein expression well retained across different tumour organoid type versus originating material
- Genetic diversity of cancer is represented
What are future opportunities for PDOs to model patient responses in the clinic?
PDO generation from an individual patient
Compare against sequence of patient tumour
Drug screen in patient-derived model to identify effective treatments
What is an example of a Co-clinical trial with PDOs?
Able to derive from cells from metastatic colon cancer patients pre and post treatment with chemotherapy Paclitaxel, able to establish an organoid that was responsive to Paclitaxel, therapy, whereas when attained patient tumour from the resistant metastatic biopsy, found that the organoid was resistant.
What are disadvantages of PDOs for modeling patient tumours?
• Protocols currently support models that are epithelial
– Lack other cell types present in the tumour micro- environment
– Not suitable for immunotherapy testing
– Co-culturing methods possible but require further development
- Derivation of model can vary depending on source tissue and quality of initial sampling
- Overgrowth with normal epithelial tissue in culture can be an issue with some models e.g. prostate PDOs
- Need rigorous testing before applicable for pre-clinical drug discovery
What are the three in vivo models in cancer?
- Genetically engineered mouse (GEM) models
- In vivo cell line models
- Patient Derived Xenograft (PDX) models
How are genetically engineered mouse (GEM) models formed?
Generated through introduction of genetic mutations associated with cancer of interest
Involves:
- Embryo editing
- Gain of function (oncogenes)/ loss of function (tumour suppressors)
- Knock-in/ Knock-out
- Ubiquitous or site specific (tissue specific promoter)
- Constitutively or conditionally expressed (inducible)
- Result in development of spontaneous tumours
What are the pros of using genetically engineered mouse models?
- Tumours occur in the relevant in situ environment
- Tumours are heterogeneous and polyclonal
- Intact immune system – allows modelling of tumour microenvironment
What are the cons of using genetically engineered mouse models?
• Models slow to develop – can take several years
What are two types of In vivo cell line models?
Syngeneic model – mouse cell line in immunocompetent mice
Xenograft model – human cell line in immunocompromised mice - - Genetically modified immune system so that human cells are not rejected
What are three Implantation Sites in an in vivo cell line models?
- Ectopic – subcutaneous injection
- Orthotopic – site specific e.g. specific organ
- Metastatic model – injection into bloodstream
What are the pros of in vivo cell line models?
- Well characterized cell lines
- Can screen drug candidates in vitro before moving in vivo
- Easy to obtain large numbers of cells for in vivo experiments
What are the cons of in vivo cell line models?
- Do not capture the heterogeneity of human tumours
- Long term growth in vitro can result in aberrant selection pressures
- Often derived from highly aggressive metastatic tumours – less useful for modelling events in evolution of primary tumour
What are in vivo cell line models?
Cell lines originally derived from patient tumours and immortalised to continuously grow in 2d cell cultures. Can then be transplanted into mouse hosts.
What are Patient-Derived Xenograft (PDX) models
Implantation of patient tumour into immunocompromised mice
Serially passaged through further mice to generate banks of material
PDX models can be established using tumours from which sample types?
- Primary tumour/ Metastatic site
- Surgical sample/ Biopsy/ Pleural effusion
- Single Cells/ Tumour fragments
As cell models, where can PDX samples be implanted?
Ectopic – subcutaneous injection
Orthotopic – site specific e.g. specific organ
What are was found when metastatic Colorectal Cancer PDXs: Response to cetuximab (EGFR Ab) treatment was investigated?
Metastatic Colorectal Cancer PDXs: Response to cetuximab (EGFR Ab) treatment:
Using PDX models found that a large proportion of KRAS WT PDXs that did not respond to cetuximab had HER2 amplification
Dual targeting of HER2 and EGFR induced tumor regression in PDXs from KRAS WT, HER2 amplified, cetuximab resistant patients
Drugs: Pertuzumab – mAB, disrupts HER2 dimerization with EGFR
Lapatinib – small molecule inhibitor or HER2 + EGFR
PDX models can be used as avatars to predict patient responses to treatment
What is an example?
Irinotecan, cetuximab and bevacizumab resulted in marked decrease in tumour volume in PDX
Treatment of patient with irinotecan, cetuximab and bevacizumab resulted in marked decrease in tumour volume
What are limitations of PDX models in cancer research?
- Rate of engraftment of PDX models can vary
- Mice are immunocompromised = loss of interaction between tumour and immunological component
- Expensive and labour intensive compared to cell line models
- Rigorous pre-clinical testing involves high numbers of mice = limits of large-scale testing.
- [Gao et al (2015) have suggested this could be circumvented using a ‘1 PDX per patient model per treatment’ approach]
Tumour biology can be modelled using what 3 pre-clinical laboratory cancer models?
– 2D cancer cell line cultures
– Patient derived organoids “PDO”
– Patient derived xenografts “PDX”
